Ionotropic glutamate receptors are the predominant mediators of excitatory synaptic signals in the mammalian central nervous system. Glutamate binding triggers the formation of transmembrane ion channels in the receptor protein, permitting cations to flow down the resultant electrochemical gradients and across the postsynaptic membrane, thus depolarizing it, and thereby stimulating the receiving cell. In addition to synaptic transmission, the glutamate receptors play an important role in the regulation of synaptic strength and in diverse neuropathologies, including epilepsy and stroke (reviewed in 1). According to agonist affinity profiles, these receptors can be subdivided into three subfamilies: α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors, N-methyl-D-aspartate (NMDA) receptors, and kainate receptors (1, 2). Because there is significant cross-reactivity between the ligands that activate the AMPA and kainate receptors, these two receptor types are grouped together under the term “non-NMDA glutamate” receptors (3–5).
Antagonists of all the three subtypes of the ionotropic glutamate receptor have been found to have protective effects against both chronic and acute neurodegenerative processes in animal models (6–11). Most of the current research has been focused on the NMDA antagonists. Antagonists of non-NMDA glutamate receptors have not been explored to the same extent.
At present, two methods are conventionally used for screening for compounds that bind to receptor proteins such as glutamate receptors, namely radioactive ligand binding (12) and electrophysiological current recording measurements (13). In the radioactive ligand binding method, competitive displacement of a radioactive ligand (such as 3H-AMPA) by the test compound is used as the basis for determining the affinity of the test compound to the receptor. In the electrophysiological method, the function of the compound in activating or deactivating the receptor (as measured by the ionic currents mediated by the receptor) is used as the basis for determining the affinity of the test compound to the receptor. As a general proposition, radioactive ligand binding is the more commonly used method because it is less time consuming relative to electrophysiological approach. There remains, however, a long-felt and unmet need for a ligand-binding assay that is as fast or faster than the radioactive ligand binding method (while maintaining comparable accuracy and precision), but that does not require the use of radioactive reagents. Such an alternative is the subject of the present invention.